Crude drug extracts, and methods for making and standardizing same

ABSTRACT

Crude drug extracts containing soluble silicon compounds as an effective component are obtained by subjecting a crude drug to extraction with water or an aqueous solvent, preferably at an alkaline pH. The crude drugs subjected to extraction to obtain the extracts may be derived from animals, plants, etc. The quality of the crude drug extract can be standardized using the soluble silicon compounds as an index. The soluble silicon compounds exhibit inhibitory action towards the production of plasma kallikrein. The amount of soluble silicon compounds in the dry extract may be used as an index whereby the quality of various crude drugs can be standardized which contributes to providing crude drug extracts having a stable or consistent quality. Consequently, the present invention greatly contributes to the appropriate standardization of pharmaceuticals.

This is a divisional of application Ser. No. 09/200,918 filed Nov. 30,1998.

FIELD OF THE INVENTION

The present invention relates to crude drug extracts containing solublesilicon compounds as an effective component, methods for making thecrude drug extracts, and also to standardization methods for crude drugsand their extracts.

BACKGROUND OF THE INVENTION

Living organisms conducting living phenomenon are composed of cellswhere functional abnormality in the cells introduces the livingorganisms to a diseased state. Living organisms survive as an individualby adjusting and maintaining their physical and chemical states to andwithin certain stable physiological conditions corresponding to changesin internal and external circumstances. It is well known that themaintenance and the normalization of the biofunction are especiallycarried out by various receptors on cell surfaces and the ion channelssuch as sodium, potassium, calcium, etc. However, if the above-mentionedbiofunctions are unbalanced for some reason and it becomes chronic,so-called morbidity results causing various diseases.

The cell membrane consists of lipid bilayers and has an important andcomplicated function for maintenance of life such as selectivepermeability, active transport, generation of bioelectricity, expressionof immunoactivity, etc. Although normal cells have fluidity and exhibitself-repairing ability to injury, the fluidity of the cell membranedecreases due to internal and external invasions. Exemplary of suchinvasions are aging, various diseases, and excessive stress stimulationincluding viral and bacterial infection. The invasions and consequentdecrease in fluidity deteriorates maintenance of the homeostasis of aliving body. For example, it is well known that vascular endothelialcells and nerve cells are injured by hyperlipemia, hypertension,diabetes mellitus, aging, smoking, etc. resulting in arteriosclerosis,renal diseases, peripheral nervous disorder, etc.

A known mechanism for adjusting the complicated functions in vivo, is anenzymatic system called the kallikrein-kinin system. With respect tothis plasma kallikrein-kinin system, it is believed that a bloodcoagulation factor XII (a Hageman factor, abbreviated as FXII) isactivated due to stimulation by a lesion or an invasion to the tissuesin vivo whereby a series of enzymatic reactions is induced. Thus, theactivated blood coagulation factor XII (abbreviated as FXIIa) acts onplasma prekallikrein which exists in the same plasma to convert it toplasma kallikrein which is an enzyme in activated form. Then, the plasmakallikrein acts on high-molecular-weight kininogen (abbreviated as HK)in the plasma to liberate bradykinin.

The bradykinin which is a product of the plasma kallikrein-kinin systemexhibits various physiological activities such as dilation of peripheralblood vessels, acceleration of permeation of blood vessels, induction ofpain, generation of inflammation, migration of leucocytes, etc.Bradykinin has also been known as a mediator for induction of pain,inflammation and allergic reactions. Accordingly, when an excessiveliberation and production of bradykinin is inhibited, it is possible torelieve pain, inflammation, allergic syndromes, etc. and to make suchunhealthy states normal.

The plasma kallikrein-kinin system acts in vivo having a closerelationship with various other enzymatic reaction systems such as therenin-angiotensin system, the blood clotting system, the fibrinolysissystem, the complement system as well as the catecholamine andarachidoic acid cascade mainly related to prostaglandins, leukotrienesand thromboxanes. Accordingly, the kallikrein-kinin system is closelyrelated to blood pressure regulating action, action through the bloodclotting-fibrinolysis-complement system or bioregulation and improvingaction for peripheral circulation by various physiologically activesubstances produced by the arachidoic acid cascade and plays animportant role in the regulation of functions in vivo. Thus, the plasmakinin-kallikrein system basically relates to biofunctions andparticipates in various bioregulation systems. Therefore, it has beensuggested that a substance having an effect on the plasmakinin-kallikrein system shows various pharmacological activities.

The present inventor has conducted a study, where a plasmakallikrein-kinin system is utilized, paying attention to siliconcompounds which regulate immune action and autonomic action of nervecells in living organisms. Silicon is widely distributed in the animaland vegetable kingdoms. Especially in animal tissues, it is abundantlypresent as silicic acid in skin, hair, bone and various organs such asthe lung, adrenal gland, thymus, pancreas and spleen. Silicon is knownto be an essential component for the formation of bones. Further, inanimal tissues, silicic acid forms a collagen cross-linking chain and iscontained as a constituting component of acidic mucopolysaccharides andit has been suggested that elasticity of skin is related to the amountof silicic acid.

Crude drugs have been used in medical treatment since ancient time buttheir quality has been mostly confirmed only by means of empiricalextracting methods for many years. For example, in the methods ofevaluating the quality of crude drugs in the Japanese Pharmacopoeia(13th Edition), many of the methods are merely a confirmation test bymeans of color reaction or spots in thin layer chromatography. Undersuch circumstances, there has been a strong demand for establishing astandardization for the substances so that a predetermined effect forthe crude drug can be guaranteed. Standardization for quality of variouscrude drugs by means of a substantial index related to pharmaceuticaleffect would contribute to providing crude drug extracts having a stablequality and would greatly contribute to an appropriate standardizationof pharmaceuticals.

The present inventor has studied silicon compounds in living organismsand regulated the quality of crude drugs using silicon compounds toexpress their pharmaceutical effect in vivo whereupon the presentinvention has been accomplished. The present invention provides crudedrug extracts containing soluble silicon compounds as an effectivecomponent which exhibit inhibitory action against the production ofplasma kallikrein. The present invention also provides methods formanufacturing crude drug extracts, and standardizing methods for crudedrugs and extracts thereof.

SUMMARY OF THE INVENTION

A crude drug extract which inhibits production of plasma kallikrein maybe produced by subjecting a plant or animal crude drug to extractionwith water or an aqueous-based extracting solvent to obtain an extracthaving at least about 0.05 mg of at least one soluble silicon compoundcalculated as silicon per gram of dry extract, as an effectivecomponent. In preferred embodiments, an alkaline extracting solvent isemployed to substantially increase the soluble silicon compound contentof the crude drug extract. The extracting solvent may have a pH of fromabout 8.5 to about 10.5. The reaction may be conducted at roomtemperature, or the extract may be heated or boiled, followed byevaporating the solvent therefrom to give a dry substance. Examples ofaqueous-based extraction solvents include mixtures of water with ethanolor phenol. In embodiments of the invention, prior to extraction with thealkaline extracting solvent, the crude drug may be subjected toextraction with an extracting solvent having a neutral pH. The crudedrug extracts of the present invention may be used in pharmaceuticallyeffective amounts as an anti-allergic agent, an analgesic agent, or ananti-inflammatory agent to treat patients in need thereof.

Standardizing or evaluating animal or plant crude drugs forpharmaceutical effectiveness may be achieved by subjecting an animal orplant crude drug to extraction to obtain an extract solution, drying theextract solution to obtain a dry crude drug extract, determining theamount of soluble silicon compounds calculated as silicon per gram ofdry crude drug extract, and comparing the determined amount of solublesilicon compounds to a minimal amount needed to obtain inhibition ofplasma kallikrein production. In embodiments of the invention,standardizing crude drug extracts, such as plant crude drug extracts maybe performed by determining the amount of soluble silicon compoundscalculated as silicon per gram of dry crude drug extract, measuringinhibiting action of the crude drug extract against plasma kallikrein,and subjecting the crude drug extract to a plurality of coloringreactions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to crude drug extracts which contain notless than 0.05 mg of soluble silicon compounds calculated as silicon pergram of the dry crude drug. The crude drug extract of the presentinvention may be obtained by extraction of various crude drugs, forexample, animal and plant crude drugs including tanjin (Salviaemilitiorrhizae radix), shireishi (Ganoderma lucidum), creeping saxifrage(Saxifraga stolonifera), scouring rush (Equisetum hiemale), Chinesegutta percha, plantago herb, plantago seeds, chorei (polyporussclerotium), saiko (bupleurum root), Japanese angelica root, elderberry,bukuryo (poria sclerotium), pueraria root, crude aloe, ginseng, ginger,alisma rhizome, schisandra fruit, sanshiti (root of Panax nothoginseng),jujube, chinpi (citrus unshiu peel), bakumondo (ophiopogon tuber), youngstaghom, oriental bezoar, lumbicusa, bear bile, longgu, etc. The presentinvention is applicable to any crude drug extract derived from animals,plants, minerals, etc. satisfying the requirements of the presentinvention. Such crude drug materials are extracted with water, ethanolor a suitable extracting solvent to which an additive such as phenol isadded whereby the crude drug extract can be manufactured. At that time,extraction and concentration of the active substances may be enhanced byheating or changing the pH of the solvent. Thus, the followingmanufacturing methods may be exemplified for obtaining crude drugextracts in accordance with present invention:

1) Pure water is added to a crude drug material, the mixture is boiledwith stirring and the insoluble matters are removed by filtration or thelike to give an extract solution. The extract solution is concentratedif necessary and spray-dried or freeze-dried in vacuo to give a powder.

2) Pure water is added to a crude drug material, the mixture is boiledwith stirring and the insoluble matters are removed by filtration or thelike to obtain an extract. Pure water is further added to the residue,pH is adjusted to an alkaline region (to 8.5-10.5, for example to around9.5), then the mixture is boiled with stirring again, and the insolublematters are removed by filtration or the like to obtain an extract, andthe extract is adjusted to about the neutral pH region and combined withthe already-prepared first extract. Then, the combined extract isconcentrated and/or evaporated to dryness if necessary to give a powder.Spray-drying or freeze-drying in vacuo to give a powder may be employedas in the above-mentioned method 1).

3) To a crude drug material is added a 1% aqueous phenol solutionfollowed by boiling with stirring and the insoluble matters are removedby filtration or the like to give an extract solution. The extractsolution is concentrated if necessary and spray-dried or freeze-dried invacuo to give a powder.

4) Pure water and ethanol are added to a crude drug material, themixture is boiled with stirring and the insoluble matters are removed byfiltration or the like to give an extract solution. The extract solutionis concentrated if necessary and spray-dried or freeze-dried in vacuo togive a powder.

5) After conducting the extracting operations as described in the abovemethods 1) to 4), the pH of the extract is adjusted to weakly alkaline(for example, to pH of around 8.5) followed by concentrating, and the pHof the concentrate is adjusted to nearly the neutral region followed bypulverizing in the same manner as mentioned above.

Conventional pH adjusting agents, such as inorganic or organic bases andacids and salts may be employed to obtain a desired pH for theextracting solvent and extract. For example, alkali metal hydroxidessuch as sodium hydroxide, and potassium hydroxide, etc. may be employedto obtain a desired alkaline pH. Exemplary acids which may be employedto adjust pH to the neutral range include hydrochloric acid, sulfuricacid, and hydrobromic acid, etc.

In accordance with the present invention, crude drug extracts arecharacterized and evaluated for pharmaceutical effectiveness byspecifying the soluble silicon compound content of the crude drugextract. The content of the soluble silicon compounds in the dried crudedrug extract obtained by the above-mentioned manufacturing methods canbe analyzed by the following method and is able to be regulated as anamount calculated as silicon.

Thus, the crude drug extract is added to water (to an extent of 1mg/ml), the mixture is subjected to stirring and an ultrasonictreatment. In preferred embodiments, the stirring is conducted at roomtemperature for about ten minutes and ultrasonic treatment is conductedat room temperature for about ten minutes. Then, the insoluble mattersare removed by filtration or centrifugation, and the amount of siliconin the resulting solution is measured by a molybdenum blue method. Inaddition, an inhibiting action of the same sample solution against theproduction of plasma kallikrein is measured and is confirmed as an indexfor the soluble silicon compounds. The measured inhibiting actionagainst plasma kallikrein production is important as an index for themeasurement and confirmation of the titer (potency of the biologicalactivity) of the soluble silicon compounds having a biological activity.

In addition to the crude drug extract in which the soluble siliconcompounds are specified, the present invention further covers variousembodiments such as a method of manufacturing the same and itspharmaceutical use. Preferred embodiments of the present invention are:

(1) A crude drug extract containing 0.05 mg or more soluble siliconcompounds (calculated as silicon) per gram of the dry substance.

(2) A crude drug extract according to the above paragraph (1) which isextracted from one of the animal and plant crude drugs selected from thegroup consisting of tanjin (Salvia militiorrhiza radix), shireishi(Ganoderma lucidum), creeping saxifrage (Saxifraga stolonifera),scouring rush (Equisetum hiemale), Chinese gutta percha, plantago herb,plantago seeds, chorei (polyporus sclerotium), saiko (bupleurum root),Japanese angelica root, elderberry, bukuryo (poria sclerotium), puerariaroot, crude aloe, ginseng, ginger, alisma rhizome, schisandra fruit,sanshiti (root of Panax nothoginseng), jujube, chinpi (citrus unshiupeel), bakumondo (ophiopogon tuber), young staghorn, oriental bezoar,lumbicusa, bear bile, longgu, etc.

(3) A crude drug extract according to the above paragraph (1) which isextracted from one of the crude drugs selected from the group consistingof tanjin (Salvia militiorrhiza radix), shireishi (Ganoderma lucidum),creeping saxifrage (Saxifraga stolonifera), scouring rush (Equisetumhiemale), Chinese gutta percha, plantago herb, plantago seeds, chorei(polyporus sclerotium), saiko (bupleurum root), Japanese angelica root,elderberry, bukuryo (poria sclerotium), pueraria root, crude aloe andginseng.

(4) A crude extract according to the above paragraph (1), (2) or (3) inwhich water is added to the dry substance (to an extent of 1 mg/mL) andthe amount of the silicon compounds existing in a solubilized state insaid aqueous solution is regulated or specified.

(5) A method for the manufacture of a crude drug extract according toany one of the above paragraphs (1) to (4) in which an extractingsolvent where the pH is adjusted to an alkaline region is used.

(6) A manufacturing method according to the above paragraph (5) in whichan extracting solvent where the pH is adjusted to from 8.5 to 10.5 isused.

(7) A method for the manufacture of a crude drug extract according toany one of the above paragraphs (1) to (4) in which an extraction isconducted using an extracting solvent near the neutral pH region (pH ofabout 7), followed by using an extracting solvent where the pH isadjusted to an alkaline region, such as a pH of from 8.5 to 10.5.

(8) A manufacturing method according to any of the above paragraphs (5)to (7) in which the crude drug material is extracted by heating orboiling followed by evaporating the solvent therefrom to give a drysubstance.

(9) A manufacturing method according to any of the above paragraphs (5)to (8) in which water, ethanol or a mixed solution thereof is used as anextracting solvent.

(10) A manufacturing method according to the above paragraph (9) inwhich an extracting solvent to which an additive such as phenol is addedis employed.

(11) A method for the standardization of a crude drug extract in whichsoluble silicon compounds are used as an index.

(12) A method according to the above paragraph (9) in which a crude drugmentioned in paragraph (2) or (3) is standardized.

(13) A method according to the above paragraph (12) in which the crudedrug is standardized by a method according to paragraph (4) above.

(14) A method according to any of the above paragraphs (11) to (13) inwhich the crude drug is standardized by combining identifying tests suchas a coloring reaction.

(15) An inhibiting agent against plasma kallikrein production containinga crude extract according to any of paragraphs (1) to (4) above as aneffective component.

(16) An inhibiting agent against plasma kallikrein production accordingto the above paragraph (15) in which said agent is an anti-allergicagent.

(17) An inhibiting agent against plasma kallikrein production accordingto the above paragraph (15) in which said agent is an analgesic agent.

(18) An inhibiting agent against plasma kallikrein production accordingto the above paragraph (15) in which said agent is an anti-inflammatoryagent.

An extract of a crude drug of the present invention can be used as adrug material in extract form or a dried powder form. The crude drugextract can be made into pharmaceutical preparations as is without anyexcipients or together with commonly-used excipients. For example, acrude drug extract which is an effective component of the pharmaceuticalcompositions of the present invention can be made into variouspharmaceutical compositions or preparations by combining one or more ofthe extracts with at least one pharmaceutical carrier or diluent. Theextracts can be made into various types of preparations by knownmethods. The pharmaceutical preparations or compositions may be madeinto solid, semi-solid, liquid or aerosol formulations for oraladministration (e.g. tablets, capsules, powders, liquids, etc.) and forparenteral administration (e.g. for subcutaneous, intravenous,intramuscular, intrarectal and intranasal administrations).

The extracts of the present invention may be used either solely orjointly in pharmaceutically effective amounts for treating animals orhumans. They may also be used in pharmaceutically effective amounts incombination with pharmaceutically effective amounts of otherpharmaceutically active components in pharmaceutical compositions orpreparations.

In the case of preparations for oral administration, one or more of theextracts of the present invention alone or together with commonly-usedpharmaceutically acceptable excipients in pharmaceutically acceptableamounts. For example, for oral administration, the extract can be usedas a pharmaceutical preparation preferably as is or together withbuffers, preservatives, flavors such as saccharides, perfumes, etc. Whenmaking the extract dry and into a powder form, the dried powder as is ortogether with commonly-used excipients such as a suitablepharmaceutically acceptable additive or carrier (e.g. lactose, mannitol,corn starch, potato starch, etc.) may be mixed with one or morepharmaceutically acceptable: (1) binders such as crystalline cellulose,cellulose, cellulose derivatives, gum arabicum, corn starch, gelatin,etc., (2) disintegrating agents such as corn starch, potato starch,potassium carboxymethyl-cellulose, etc., (3) lubricating agents such astalc, magnesium stearate, etc., and (4) other pharmaceuticallyacceptable excipients including pharmaceutically acceptable bulkingagents, moisturizing agents, buffers, preservatives, perfumes and thelike to obtain tablets, diluted powders, granules or capsules.

In the case of parenteral administration using injections, for example,it is possible to prepare solutions or suspensions of one or moreextracts of the present invention in pharmaceutically acceptablecarriers such as aqueous and nonaqueous solvents such as distilled waterfor injection, physiological saline solution, Ringer's solution, plantoil, synthetic fatty acid glycerides, higher fatty acid esters,propylene glycol, etc.

It is also possible, depending upon the type of the disease, to preparepharmaceutical preparations other than the above-mentioned ones such assuppositories, inhalations, aerosol preparations, collyriums, ointments,poultices, etc.

For example, suppositories may be prepared by mixing at least oneextract of the present invention with pharmaceutically acceptableamounts of one or more pharmaceutically acceptable fatty/oily bases(e.g. cacao butter), emulsified bases, water-soluble bases (e.g.Macrogol), hydrophilic bases, etc.

In the case of inhalations or aerosol preparations, at least one extractof the present invention in the form of a liquid or minute powder can befilled up in an aerosol container with a gas or liquid spraying agent,and if desired, with conventional adjuvants such as one or morepharmaceutically acceptable humidifying agents or dispersing agents.They can also be used as pharmaceuticals for a non-pressurizedpreparation such as in a nebulizer or an atomizer.

In order to make the extracts of the present invention into collyriums,they can be prepared as a solution or suspension together with anaqueous solvent such as sterile, purified water and physiologicallysaline solution, or a non-aqueous solvent for injection. The collyriumsmay also include pharmaceutically acceptable preservants, sterilizingagents, pH adjusting agents, and the like.

The present invention will now be further illustrated by way of thefollowing non-limiting examples wherein all parts, percentages, andratios are by weight, all temperatures are in ° C. or room temperature,and all pressures are in psia or atmospheric unless otherwise indicated:

EXAMPLE A Standardization Tests

Standardization tests used to characterize and evaluate thepharmaceutical effectiveness of extracts of crude drugs in accordancewith the present invention are a Quantitative Test, an Activity Test,and Confirmation Tests:

I. Quantitative Determination of the Soluble Silicon Compounds(“Quantitative Test”)

Water was added to dry powder of a crude drug extract (to an extent of 1mg/mL), the mixture was stirred at room temperature for ten minutes,subjected to an ultrasonic treatment at room temperature for ten minutesand filtered through a membrane filter (0.45 μm) to remove the insolublematters, and the filtrate was used as a sample solution to be tested. To3 mL of this sample solution was added 0.2 mL of sodium hydroxidesolution (1 mol/L), the mixture was allowed to stand overnight and 0.3mL of hydrochloric acid (1 mol/L) was added thereto. After that, 0.1 mLof ammonium molybdate solution (prepared by thorough mixing of 15 g ofammonium molybdate tetrahydrate with 64 mL of a ten-fold diluted aqueoussolution of hydrochloric acid to dissolve it, followed by adding waterthereto to make 200 mL) was added thereto and mixed therewith. Themixture was allowed to stand for five minutes, mixed with 0.4 mL of anaqueous solution of tartaric acid (170 g/L) and, after one minute,colored by mixing with 0.1 mL of a 1-amino-2-naphthol-4-sulfonic acidsolution (prepared by dissolving 1.4 g of sodium sulfite anhydride, 0.3g of 1-amino-2-naphthol-4-sulfonic acid and 18 g of sodium hydrogensulfite in water to make 200 mL). After allowing to stand for about 30minutes, the absorbance at 820 nm wave length was measured so thatquantitative determination of silicon was conducted. In the followingExamples, the result (μg/mL) is given under the abbreviation“Quantitative Test”.

II. Inhibiting Action Against Plasma Kallikrein Production (“ActivityTest”)

Inhibiting action of the crude drug extract of the present inventionagainst the production of plasma kallikrein was measured according to amethod described in the literature [Kiso to Rinsho, volume 20, no. 17,pages 399-405 (1986)].

Thus, a kaolin suspension was added to normal human plasma diluted witha physiological saline solution. After a certain time, lima bean trypsininhibitor was added to cease the kallikrein production reaction. Afterthat, the substance to be tested is made to coexist in the system wherethe resulting kallikrein was to be measured using a synthetic substrate(D-Pro-Phe-Arg-p-nitroaniline) whereby the inhibiting activity of saidsubstance to be tested against the kallikrein production was determined.Absorbance at 405 nm was measured using the amount of p-nitroanilineliberated from the synthetic substrate by the produced kallikrein as anindex. The potency of the inhibiting activity was expressed as thedifference between the absorbance where the substance to be tested wasadded as above and that where it was not added (control). Thus, thegreater the difference (ΔOD) between the absorbances, the greater is theinhibiting activity against the production of kallikrein. When thesubstance to be tested had a very strong inhibiting activity, the testwas conducted using a sample solution where the above test sample (1mg/mL) was diluted to an appropriate extent. Also, the result calculatedfor the activity potency of the original sample concentration based uponthe degree of dilution was given. In the following examples, the result(ΔOD) is given under the abbreviation “Activity Test”.

III. Confirmation Tests of the Components Contained in the Crude DrugExtract (“Confirmation Tests” 1 through 13)

Test (1): Detection of Pentoses (by an orcinol-ferric chloride method)

Sample solution: Crude drug extract (0.1 g) was dissolved in 10 mL ofwater.

Standard solution: D-Ribose (0.1 mg) was dissolved in 1 mL of water.

Operation: To a sample solution and 1 mL of a standard solution wereadded 3 mL of a ferric chloride solution and 0.3 mL of an ethanolicsolution of orcinol followed by thorough stirring. When the mixture washeated for 25 minutes on a water bath and cooled in running water, thesolution gave a green color.

Coloring reagents: a) Ferric chloride (0.1 mg) was dissolved in 100 mLof hydrochloric acid; b) Orcinol (0.1 mg) was dissolved in 100 ml ofethanol.

Test (2): Detection of Hexoses (by an anthrone-sulfuric acid method)

Sample solution: Crude drug extract (0.1 g) was dissolved in 10 mL ofwater.

Standard solution: D-Glucose (0.1 mg) was dissolved in 1 mL of water.

Operation: Ice-cooled anthrone-sulfuric acid (5 mL) was added to andmixed with an ice-cooled sample solution and the standard solution (1mL). When the mixture was heated for ten minutes on a water bath andcooled in running water, the solution gave a green color.

Coloring solution: Anthrone (0.2 g) was dissolved in 100 mL ofice-cooled sulfuric acid and the solution was added to 20 mL of waterwith ice-cooling.

Test (3): Detection of Steroids/Saponins (by a Liebermann's reaction)

Operation: To 0.1 g of a crude drug extract was added 2 mL of aceticanhydride, the mixture was thoroughly stirred, heated for two minutes ona water bath, allowed to stand at room temperature. When 2 mL ofsulfuric acid was gently added to 0.7 mL of a supernatant aceticanhydride layer, the interface gave a red to reddish brown color or theupper layer gave a blue to green color.

Test (4): Detection of Carboxyl Group-Containing Compounds (by2,4-dinitrophenylhydrazine)

Sample solution: To 0.1 g of a crude drug extract was added 3 mL ofethanol, the mixture was thoroughly stirred and allowed to stand and thesupernatant liquid was used as a sample solution.

Standard solution: Anisaldehyde (10 mg) was dissolved in 3 mL of ananhydrous ethanol to prepare a standard solution.

Operation: A 2,4-dinitrophenylhydrazine solution (1 mL) was added to asample solution and a standard solution and, when they were mixed andallowed to stand, the mixture gave a precipitate which was yellow toorange in color.

Reagent: 2,4-Dinitrophenylhydrazine (1.5 g) was dissolved in a cooledmixture of 10 mL of sulfuric acid and 10 mL of water and then water wasadded thereto to make 100 mL.

Test (5): Detection of Phenol Group-Containing Compounds (by a ferricchloride method)

Sample solution: To 0.1 g of a crude drug extract was added 3 mL of ananhydrous ethanol, the mixture was thoroughly mixed and allowed to standand the supernatant liquid was used as a sample solution.

Operation: To 1 mL of the sample solution was added 1 mL of a dilutedferric chloride solution and, after the mixture was stirred, thesolution gave a blue color.

Reagent: Diluted, ferric chloride (9 g) was dissolved in water and thenwater was added to 2 mL of the resulting solution to make 10 mL.

Test (6): Confirmation of Flavonoids

Sample solution: To 50 mg of a crude drug extract was added 10 mL ofmethanol, the mixture was gently heated for 2-3 minutes and centrifuged,then 0.1 g of magnesium ribbons and 1 mL of hydrochloric acid were addedto the supernatant liquid and, when the mixture was allowed to stand,the liquid gave a red color.

Test (7): Aldehydes and Ketones (by 2,4-dinitrophenylhydrazine)

Sample solution: To 0.05 g of a crude drug extract was added 1 mL ofdiluted ethanol, the mixture was thoroughly mixed and allowed to stand,and the supernatant liquid was used as a sample solution.

Developing solvent: an upper layer of n-butanol-acetic acid-water(4:1:5)

Thin layer plate: silica gel (Merck 5553)

Amount of the Sample: 5 μL

Coloring reagent: 2,4-Dinitrophenylhydrazine (0.4 g) was dissolved in 2N hydrochloric acid to make 100 mL. After the sample solution wasdeveloped and the coloring reagent was sprayed thereon and allowed tostand, it gave a yellow to brown color.

Test (8): Terenes/steroids/saccharides (by anisaldehyde)

Sample solution: To 0.05 g of a crude drug extract was added 1 mL ofdiluted ethanol, the mixture was thoroughly mixed and allowed to stand,and the supernatant liquid was used as a sample solution.

Developing solvent: an upper layer of n-butanol-acetic acid-water(4:1:5)

Thin layer plate: silica gel (Merck 5553)

Amount of the Sample: 5 μL

Coloring reagent: To 0.5 mL of p-anisaldehyde was added 1 mL of sulfuricacid followed by adding ethanol thereto to make 20 mL. After the samplesolution was developed, the coloring reagent was sprayed thereon and,when heated at 105° C. for five minutes, it gave a blue to purple colorand a gray to black color.

Test (9): Amine/indole derivatives (by p-dimethylamino-benzaldehyde)

Sample solution: To 0.05 g of a crude drug extract was added 1 mL ofdiluted ethanol, the mixture was thoroughly mixed and allowed to standand the supernatant liquid was used as a sample solution.

Developing solvent: an upper layer of n-butanol-acetic acid-water(4:1:5)

Thin layer plate: silica gel (Merck 5553)

Amount of the Sample: 5 μL

Coloring reagent: p-Dimethylaminobenzaldehyde (1 g) was dissolved in 50mL of hydrochloric acid and then 50 mL of ethanol was added thereto.After developing the sample solution, the coloring solution was sprayedthereon followed by allowing it to stand whereupon it gave a blue topurple color.

Test (10): Detection of Tertiary Amines (by a Dragendorff's reagent)

Sample solution: To 0.05 g of a crude drug extract was added 1 mL ofethanol, the resulting dispersion was treated with ultrasonic waves for30 minutes and heated at 60° C. for five minutes to obtain an extract.The extract was centrifuged and the supernatant liquid was used as asample solution.

Developing solvent:

a) an upper layer of n-butanol-acetic acid-water (4:1:5)

b) methanol

Thin layer plate: silica gel 60F254

Amount of the Sample: 5 μL

Coloring reagent: prepared according to the Japanese Pharmacopoeia (13thedition).

Operation: The sample solution was developed, sprayed with a coloringsolution and allowed to stand to give a yellow to orange color.

Test (11): Alkaloids (by a platinum chloride-potassium iodide solution)

Sample solution: To 0.05 g of a crude drug extract was added 1 mL ofethanol and the resulting dispersion was treated with ultrasonic wavesfor 30 minutes and heated at 60° C. for five minutes to obtain anextract. The extract was centrifuged and the supernatant liquid was usedas a sample solution.

Developing solvent:

a) an upper layer of n-butanol-acetic acid-water (4:1:5)

b) methanol

Thin layer plate: silica gel 60F254

Amount of the Sample: 5 μL

Coloring reagent: prepared according to the Japanese Pharmacopoeia (13thedition).

Operation: The sample solution was developed, sprayed with a coloringsolution and allowed to stand to give a reddish brown color.

Test (12): Substances Colored with Antimony Trichloride

Sample solution: To 100 mg of a crude drug extract was added 2 mL of 50%ethanol, the mixture was gently heated for 2-3 minutes and centrifugedand the supernatant liquid was used as a sample solution.

Developing solvent:

a) n-propanol-water (64:36)

b) n-hexan-ethyl acetate (3:7)

Thin layer plate: silica gel with fluorescence indicator (UV=365 nm)

Amount of the Sample: 3 μL

Coloring reagent: prepared according to the Japanese Pharmacopoeia (13thedition).

Operation: The sample solution was developed and, when the coloringsolution was sprayed thereon, a blue color resulted.

Test (13): Substances Colored with Ninhydrin

Sample solution: A crude drug extract (0.1 g) was dissolved in 10 mL ofwater.

Developing solvent: an upper layer of n-butanol-acetic acid-water(4:1:5)

Thin layer plate: silica gel (Merck 5553)

Amount of the Sample: 5 μL

Coloring reagent: prepared according to the Japanese Pharmacopoeia (13thedition).

Operation: The sample solution was developed and, when the coloringsolution was sprayed thereon, a blue to purple color resulted.

EXAMPLE 1

In this example, four different manufacturing or extraction methods asdescribed above were used to prepare four crude drug extracts which werethen characterized and evaluated in accordance with the presentinvention:

1) A crude drug extract was prepared according to manufacturing orextraction method 1 above. Thus, to 150 g of shireishi (Ganodermalucidum) was added 3 L of pure water, the mixture was boiled withstirring to obtain an extract, the extract was concentrated in vacuo toabout 800 mL and the concentrate was pulverized by spray-drying.

(Quantitative Test) 0.650 μg/mL

(Activity Test) 0.772

(Confirmation Test) positive for (3), (4), (7), (8), (9), (10), (11),(12) and (13)

2) A crude drug extract was prepared according to manufacturing orextraction method 2 above. Thus, to 150 g of shireishi (Ganodermalucidum) was added 3 L of pure water and the mixture was boiled withstirring to obtain an extract. To the residue was added 3 L of purewater, the mixture was adjusted to pH 9.5 and boiled with stirring forone hour to extract, the extract was adjusted to pH 7.0, combined withthe previously-prepared extract, the combined extract was concentratedin vacuo to about 800 mL and the concentrate was pulverized byspray-drying.

(Quantitative Test) 0.737 μg/mL

(Activity Test) 1.440

(Confirmation Test) positive for (3), (4), (7), (8), (9), (10), (11),(12) and (13)

3) A crude drug extract was prepared according to manufacturing orextraction method 3 above. Thus, to 150 g of shireishi (Ganodermalucidum) was added 3 L of an aqueous phenol, the mixture was boiled withstirring for one hour to obtain an extract, the extract was concentratedin vacuo to about 800 mL and the concentrate was pulverized byspray-drying.

(Quantitative Test) 0.682 μg/mL

(Activity Test) 0.608

(Confirmation Test) positive for (3), (4), (7), (8), (9), (10), (11),(12) and (13)

4) A crude drug extract was prepared according to manufacturing orextraction methods 5 and 3 above. Thus, to 150 g of shireishi (Ganodermalucidum) was added 3 L of an aqueous phenol, the mixture was boiled withstirring for one hour to obtain an extract, the extract was adjusted topH 8.5 and concentrated in vacuo to about 800 mL and the concentrate wasadjusted to 7.0 and pulverized by spray-drying.

(Quantitative Test) 0.627 μg/mL

(Activity Test) 0.824

(Confirmation Test) positive for (3), (4), (7), (8), (9), (10), (11),(12) and (13)

EXAMPLE 2

A crude drug extract was prepared according to manufacturing orextraction methods 5 and 1 above. Thus, to 150 g of creeping saxifrage(Saxifraga stolonifera) was added 3 L of pure water and the mixture wasboiled with stirring for one hour to obtain an extract. The extract wasadjusted to pH 8.5 and concentrated in vacuo to about 800 mL and theconcentrate was adjusted to pH 7.0 and pulverized by spray-drying.

(Quantitative Test) 0.388 μg/mL

(Activity Test) 1.184

(Confirmation Test) positive for (1), (2), (3), (6), (8), (9), (10),(11), (12) and (13)

EXAMPLE 3

1) The same operations of extraction, concentration and drying as inmethod 1) of Example 1 were conducted for tanjin (Salviae militiorrhizaeradix)as a starting crude drug.

(Quantitative Test) 0.304 μg/mL

(Activity Test) 1.848

(Confirmation Test) positive for (2), (5), (7), (8), (9), (10), (11),(12) and (13)

2) The same operations of extraction, concentration and drying as inmethod 2) of Example 1 were conducted for tanjin (Salviae militiorrhizaeradix) as a starting crude drug.

(Quantitative Test) 0.347 μg/mL

(Activity Test) 2.268

(Confirmation Test) positive for (2), (3), (5), (7), (8), (9), (10),(11), (12) and (13)

EXAMPLE 4

1) The same operations of extraction, concentration and drying as inmethod 1) of Example 1 were conducted for scouring rush (Equisetumhiemale) as a starting crude drug.

(Quantitative Test) 2.700 μg/mL

(Activity Test) 0.716

(Confirmation Test) positive for (2), (6), (8), (10), (11), (12) and(13)

2) The same operations of extraction, concentration and drying as inmethod 2) of Example 1 were conducted for scouring rush (Equisetumhiemale) as a starting crude drug.

(Quantitative Test) 2.728 μg/mL

(Activity Test) 0.876

(Confirmation Test) positive for (2), (6), (8), (9), (10), (11), (12)and (13)

3) The same operations of extraction, concentration and drying as inExample 2 were conducted for scouring rush (Equisetum hiemale) as astarting crude drug.

(Quantitative Test) 15.93 μg/mL

(Activity Test) 0.996

(Confirmation Test) positive for (2), (6), (8), (9), (10), (11), (12)and (13)

EXAMPLE 5

1) The same operations of extraction, concentration and drying as inmethod 1) of Example 1 were conducted for Chinese gutta percha as astarting crude drug.

(Quantitative Test) 1.276 μg/mL

(Activity Test) 0.688

(Confirmation Test) positive for (2), (4), (7), (8), (9), (10), (11),(12) and (13)

2) The same operations of extraction, concentration and drying as inmethod 2) of Example 1 were conducted for scouring rush (Equisetumhiemale) as a starting crude drug.

(Quantitative Test) 1.805 μg/mL

(Activity Test) 0.880

(Confirmation Test) positive for (2), (3), (4), (7), (8), (9), (10),(11), (12) and (13)

EXAMPLE 6

The same operations of extraction, concentration and drying as inExample 2 were conducted for plantago herb as a starting crude drug.

(Quantitative Test) 0.809 μg/mL

(Activity Test) 0.780

(Confirmation Test) positive for (2), (3), (7), (8), (10), (11), (12)and (13)

EXAMPLE 7

The same operations of extraction, concentration and drying as in method2) of Example 1 were conducted for plantago seed as a starting crudedrug.

(Quantitative Test) 0.392 μg/mL

(Activity Test) 0.752

(Confirmation Test) positive for (2), (4), (5), (7), (8), (9), (10),(11), (12) and (13)

EXAMPLE 8

1) The same operations of extraction, concentration and drying as inmethod 1) of Example 1 were conducted for chorei (polyporus sclerotium)as a starting crude drug.

(Quantitative Test) 0.725 μg/mL

(Activity Test) 0.189

(Confirmation Test) positive for (2), (3), (7), (8), (10), (12) and (13)

2) The same operations of extraction, concentration and drying as inmethod 2) of Example 1 were conducted for chorei (polyporus sclerotium)as a starting crude drug.

(Quantitative Test) 0.964 μg/mL

(Activity Test) 0.573

(Confirmation Test) positive for (2), (3), (4), (5), (8), (10), (12) and(13)

EXAMPLE 9

The same operations of extraction, concentration and drying as in method2) of Example 1 were conducted for saiko (bupleurum root) as a startingcrude drug.

(Quantitative Test) 0.281 μg/mL

(Activity Test) 0.434

(Confirmation Test) positive for (2), (3), (8), (9), (10), (11), (12)and (13)

EXAMPLE 10

The same operations of extraction, concentration and drying as in method2) of Example 1 were conducted for Japanese angelica root as a startingcrude drug.

(Quantitative Test) 0.077 μg/mL

(Activity Test) 0.164

(Confirmation Test) positive for (2), (8), (9), (10), (12) and (13)

EXAMPLE 11

1) The same operations of extraction, concentration and drying as inmethod 3) of Example 1 were conducted for elderberry as a starting crudedrug.

(Quantitative Test) 0.203 μg/mL

(Activity Test) 0.090

(Confirmation Test) positive for (2), (3), (8), (9), (10), (11), (12)and (13)

2) The same operations of extraction, concentration and drying as inmethod 4) of Example 1 were conducted for elderberry as a starting crudedrug.

(Quantitative Test) 0.227 μg/mL

(Activity Test) 0.063

(Confirmation Test) positive for (2), (3), (8), (9), (10), (11), (12)and (13)

EXAMPLE 12

1) The same operations of extraction, concentration and drying as inmethod 1) of Example 1 were conducted for bukuryo (poria sclerotium) asa starting crude drug.

(Quantitative Test) 0.969 μg/mL

(Activity Test) 0.055

(Confirmation Test) positive for (2), (3), (4), (7), (8), (12) and (13)

2) The same operations of extraction, concentration and drying as inmethod 2) of Example 1 were conducted for bukuryo (poria sclerotium) asa starting crude drug.

(Quantitative Test) 1.711 μg/mL

(Activity Test) 0.084

(Confirmation Test) positive for (2), (3), (4), (8), (10), (11), (12)and (13)

EXAMPLE 13

1) The same operations of extraction, concentration and drying as inmethod 1) of Example 1 were conducted for pueraria root as a startingcrude drug.

(Quantitative Test) 0.118 μg/mL

(Activity Test) 0.071

(Confirmation Test) positive for (2), (3), (5), (6), (8), (9), (10),(11), (12) and (13)

2) The same operations of extraction, concentration and drying as inmethod 2) of Example 1 were conducted for pueraria root as a startingcrude drug.

(Quantitative Test) 0.122 μg/mL

(Activity Test) 0.078

(Confirmation Test) positive for (2), (3), (5), (6), (8), (10), (11),(12) and (13)

EXAMPLE 14

The same operations of extraction, concentration and drying as inmethod 1) of Example 1 were conducted for crude aloe as a starting crudedrug.

(Quantitative Test) 0.536 μg/mL

(Activity Test) 0.074

(Confirmation Test) positive for (2), (5), (8), (12) and (13)

EXAMPLE 15

The same operations of extraction, concentration and drying as inmethod 1) of Example 1 were conducted for ginseng as a starting crudedrug.

(Quantitative Test) 0.087 μg/mL

(Activity Test) 0.051

(Confirmation Test) positive for (2), (3), (4), (7), (8), (10), (11),(12) and (13)

A summary of the results of analysis and evaluation of each of theextracts of Examples 1 through 15 is given in Table 1 where “p”indicates a positive result and no entry indicates a negative result forthe confirmation test:

TABLE 1 SUMMARY OF RESULTS OF ANALYSIS AND PHARMACEUTICAL ACTIVITY OFCRUDE DRUG EXTRACTS EXTRACTION QUANTITATIVE TEST ACTIVITY CONFIRMATIONTEST EXAMPLE METHOD μgSi/ml TEST 1 2 3 4 5 6 7 8 9 10 11 12 13 1-1 10.65 0.772 p p p p p p p p p 1-2 2 0.737 1.44 p p p p p p p p p 1-3 30.682 0.608 p p p p p p p p p 1-4 5(3) 0.627 0.824 p p p p p p p p p 25(1) 0.388 1.184 p p p p p p p p p p 3-1 1 0.304 1.848 p p p p p p p p p3-2 2 0.347 2.268 p p p p p p p p p p 4-1 1 2.7 0.716 p p p p p p p 4-22 2.728 0.876 p p p p p p p p 4-3 5(1) 15.93 0.996 p p p p p p p p 5-1 11.276 0.688 p p p p p p p p p 5-2 2 1.805 0.88 p p p p p p p p p p 65(1) 0.809 0.78 p p p p p p p p 7 2 0.392 0.752 p p p p p p p p p p 8-11 0.725 0.189 p p p p p p p 8-2 2 0.964 0.573 p p p p p p p p 9 2 0.2810.434 p p p p p p p p 10 2 0.077 0.164 p p p p p p 11-1 3 0.203 0.09 p pp p p p p p 11-2 5(3) 0.227 0.063 p p p p p p p p 12-1 1 0.969 0.055 p pp p p p p 12-2 2 1.711 0.084 p p p p p p p p 13-1 1 0.118 0.071 p p p pp p p p p p 13-2 2 0.122 0.078 p p p p p p p p p 14 1 0.536 0.074 p p pp p 15 1 0.087 0.051 p p p p p p p p p

It is apparent from the results of the above-mentioned Examples wherevarious crude drugs were used that, when soluble silicon compounds arecontained in more than certain amounts, the crude drug extracts exhibita pharmaceutical effect. The method of the present invention establishesa standardization for crude drugs which has been ambiguous up to now.The silicon compounds contained in the crude drug extracts includevarious kinds of compounds. In the present invention, they are whollystandardized in terms of amount calculated as silicon measured by amolybdenum blue method. Depending upon the type of the crude drug,substances therein other than the silicon compounds are varied. However,when coloring reaction tests, etc. for such substances are appropriatelyused together with the silicon content determination, more precisestandardization of crude drugs can be established.

As shown in Table 1, the Examples indicate a tendency for an increase inthe extracting efficiency of the soluble silicon compounds when theextracting operation is conducted using a solution where the pH isadjusted to an alkaline region (e.g. pH being around 9.5), as forexample in extraction method 2. Accordingly, extraction using a solutionwhere the pH is adjusted to the alkaline region (e.g. pH about 9.5) isan example of a preferred extracting method.

As such, in accordance with the present invention, when soluble siliconcompounds which provide a pharmaceutical effect, such as inhibition ofplasma kallikrein, are used as an index, the quality of various crudedrugs and extracts thereof can be standardized. Accordingly, the presentinvention achieves crude drug extracts having a stable quality andgreatly contributes to the appropriate standardization ofpharmaceuticals.

What is claimed is:
 1. A method for standardizing or evaluating plantdrugs for pharmaceutical effectiveness comprising subjecting a plantdrug to extraction to obtain an extract solution, drying the extractsolution to obtain a dry drug extract, determining the amount of solublesilicon compounds calculated as silicon per gram of dry drug extract,and comparing the determined amount of soluble silicon compounds to aminimal amount needed to obtain inhibition of plasma kallikreinproduction.
 2. A method as claimed in claim 1 wherein the plant drug issubjected to extraction with water or an aqueous solvent to obtain anextract solution and the extract solution is dried to obtain a drugextract in powder form.
 3. A method as claimed in claim 2 wherein the pHof said water or aqueous solvent for extraction is adjusted to 8.5 to10.5.
 4. A method as claimed in claim 1 wherein the amount of solublesilicon compounds is determined by a molybdenum blue method.
 5. A methodas claimed in claim 1 wherein the amount of soluble silicon isdetermined by dissolving the dry drug extract, removing insoluble matterto obtain a filtrate, admixing the filtrate with ammonium molybdatetetrahydrate, and measuring absorbance at a wavelength of 820 nm.
 6. Amethod as claimed in claim 1 wherein the standardization or evaluationfurther comprises measuring inhibiting action of the drug extractagainst plasma kallikrein production.
 7. A method as claimed in claim 1wherein the standardization or evaluation further comprises subjectingsaid drug extract to a plurality of coloring reactions.
 8. A method forstandardizing plant drug extracts comprising determining the amount ofsoluble silicon compounds calculated as silicon per gram of dry drugextract, measuring inhibiting action of the drug extract against plasmakallikrein production, and subjecting said drug extract to a pluralityof coloring reactions.
 9. A method as claimed in claim 8 wherein saidplant drug extracts are obtained by subjecting a plant drug toextraction with water or an aqueous solvent to obtain an extractsolution and the extract solution is dried to obtain a drug extract inpowder form.
 10. A method as claimed in claim 9 wherein the pH of saidwater or aqueous solvent for extraction is adjusted to 8.5 to 10.5. 11.A method as claimed in claim 8 wherein the amount of soluble silicon isdetermined by dissolving the dry drug extract in water to an extent of 1mg/mL, removing insoluble matter to obtain an aqueous solution, anddetermining the amount of the silicon in said aqueous solution.
 12. Amethod as claimed in claim 11 wherein the drug extracts are standardizedfor pharmaceutical effectiveness as an anti-allergic agent, an analgesicagent, or an anti-inflammatory agent.
 13. A method for evaluating theinhibiting activity of plant drug extracts against plasma kallikreinproduction comprising: determining the amount of soluble siliconcompounds calculated as silicon per gram of dry drug extract; andcomparing the determined amount of soluble silicon compounds to aminimal amount needed to obtain inhibition of plasma kallikreinproduction.